Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to P. gingivalis Lipopolysaccharide.

Periodontal ligament stem cells (PDLSCs) are lacking membrane CD14, which is an important component of lipopolysaccharide (LPS) signaling through toll-like receptor (TLR) 4. In the present study we investigated the effect of soluble CD14 on the response of human PDLSCs to LPS of Porphyromonas (P.) gingivalis. Human PDLSCs (hPDLSCs) were stimulated with P. gingivalis LPS in the presence or in the absence of soluble CD14 (sCD14) and the production of interleukin (IL)-6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured. The response to P. gingivalis LPS was compared with that to TLR4 agonist Escherichia coli LPS and TLR2-agonist Pam3CSK4. The response of hPDLSCs to both P. gingivalis LPS and E. coli LPS was significantly enhanced by sCD14. In the absence of sCD14, no significant difference in the hPDLSCs response to two kinds of LPS was observed. These responses were significantly lower compared to that to Pam3CSK4. In the presence of sCD14, the response of hPdLSCs to P. gingivalis LPS was markedly higher than that to E. coli LPS and comparable with that to Pam3CSK4. The response of hPdLSCs to bacterial LPS is strongly augmented by sCD14. Local levels of sCD14 could be an important factor for modulation of the host response against periodontal pathogens.

Lipopolysaccharide-binding protein inhibits toll-like receptor 2 activation by lipoteichoic acid in human odontoblast-like cells.

INTRODUCTION: Previous studies have suggested that odontoblasts sense gram-positive bacteria components through Toll-like receptor 2 (TLR2) and trigger dental pulp immunity by producing proinflammatory cytokines. Currently, the factors that modulate odontoblast TLR2 activation are unknown. Our aim was to investigate lipopolysaccharide-binding protein (LBP) effects on the TLR2-mediated odontoblast response. METHODS: Human odontoblast-like cells were stimulated with lipoteichoic acid (LTA) (a TLR2 ligand), LBP, CD14 (a TLR2 cofactor), or various combinations of LTA/LBP, LTA/CD14, or LTA/CD14/LBP. CXCL8, IL6, and TLR2 gene expression was assessed by real-time polymerase chain reaction. CXCL8 and interleukin (IL)-6 production was determined by enzyme-linked immunosorbent assay in culture supernatants of cells stimulated with LTA, LTA/CD14, or LTA/CD14/LBP. LBP effects on nuclear factor kappa B (NF-κB), p38, JNK, ERK, STAT3, and p70S6 signaling pathways were determined in LTA-stimulated odontoblast-like cells with a multiplex biometric immunoassay. LBP effects were compared with specific inhibitors of these signaling pathways. LBP transcript and protein were investigated in vivo in healthy and inflamed dental pulps by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Activation of CXCL8, IL6, and TLR2 gene expression and CXCL8 and IL-6 secretion in LTA- and LTA/CD14-stimulated odontoblast-like cells was significantly decreased by LBP. LBP inhibited NF-κB and p38 signaling pathways in LTA-stimulated cells in a similar way to NF-κB and p38 inhibitors. LBP transcript and protein were detected in vivo in inflamed dental pulps but not in healthy ones. CONCLUSIONS: These results demonstrate that LBP reduces TLR2-dependent production of inflammatory cytokines by odontoblast-like cells. We suggest that in this way it could modulate host defense in human dental pulp.

Enhanced host immune recognition of mastitis causing Escherchia coli in CD-14 transgenic mice.

Escherchia coli causes mastitis, an economically significant disease in dairy animals. E. coli endotoxin (lipopolysaccharide, LPS) when bound by host membrane proteins such as CD-14, causes release of proinflammatory cytokines recruiting neutrophils as an early, innate immune response. Excessive proinflammatory cytokines causes tissue damage, compromising mammary function. If present, soluble CD-14 (sCD-14) might out compete membrane bound CD-14, lessening the severity of the inflammatory response. To test this hypothesis transgenic mice, expressing sCD-14 in their milk (31 to 316 microg/ml), were evaluated. A cell culture study demonstrated, in the presence of LPS, milk from transgenic mice increased secretion of cytokine IL-8 compared to milk from nontransgenic littermates (18 +/- 3 vs. 35 +/- 2 ng/mL, p < 0.001). To assess protection afforded by the transgene, glands were infused with E. coli. Recovery of bacteria showed no clear relationship between sCD14 concentration and the number of organisms recovered; however, there was a strong relationship between sCD14 concentration and edema (r(2) = 0.999, p < 0.001), as measured by weight of fluid in harvested glands. Highest expressing lines had the least edema, suggesting the presence of sCD14 had an effect on reducing the inflammatory response to E. coli, thus, possibly protecting against gland tissue damage.

Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice.

AIMS: Although toll-like receptors (TLR) are known to mediate the metabolic complications of obesity, the mechanisms underlying its activation remain largely unknown. The present study analyzed a model of diet-induced obesity in mice lacking the TLR4/TLR2 co-receptor CD14. MAIN METHODS: Six-week-old male mice lacking CD14 (n = 16) were allocated to either a control diet or a high-fat high-simple carbohydrate diet (5.4 kcal/g; 35% fat; 35% sucrose), and compared with C57BL/6 (WT; n = 15) controls. After 12 weeks, body composition, basal sympathetic activity, non-invasive blood pressure and glucose tolerance were evaluated. Hepatic and adipose tissues were collected for mRNA quantification, histology and LPS incubation. KEY FINDINGS: In both WT and CD14 knockout mice, obesity was accompanied by TLR2 and TLR4 upregulation. However, obese mice lacking CD14 presented decreased lipid and macrophage content in hepatic and adipose tissues, lower urinary levels of noradrenaline, decreased systolic blood pressure, reduced fasting plasma glucose and blunted glucose intolerance, compared with obese WT group. In the presence of exogenous sCD14, adipose tissue incubation with LPS-induced TLR2 and TNF-alpha upregulation in both WT and CD14 knockout obese mice. SIGNIFICANCE: In our model of diet-induced obesity, mice lacking CD14 showed lower adiposity and hepatic steatosis, improved glucose homeostasis, blunted sympathetic overactivity and reduced blood pressure elevation. This was observed in the presence of preserved TLR4 and TLR2 gene expression, and intact TLR4 signaling pathways. These results suggest that CD14-mediated TLR activation might contribute to the cardiovascular and metabolic complications of obesity.

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts.

INTRODUCTION: Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms. METHODS: LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization. RESULTS: The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors. CONCLUSION: These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.

TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells.

CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-kappaB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products.

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.

Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

AIMS: The aim of the present study was to determine whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells). METHODS: RAW264.7 cells were treated with A. actinomycetemcomitans-lipopolysaccharide or lipopolysaccharide from Escherichia coli for 24 h. The effect of polymyxin B, l-norvaline, dl-norvaline, dexamethasone and cytokines (interferon-gamma and interleukin-4) on arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells was also determined. The cells were pretreated with anti-CD14, anti -toll-like receptor 2, or anti-toll-like receptor 4 antibody prior to stimulation with A. actinomycetemcomitans-lipopolysaccharide. Arginase activity was determined by a colorimetric assay. RESULTS: A. actinomycetemcomitans-lipopolysaccharide stimulated arginase activity in RAW264.7 cells in a dose-dependent manner, but was less potent than E. coli-lipopolysaccharide. Polymyxin B and l-norvaline, but not dl-norvaline, blocked the arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. Dexamethasone and interleukin-4 but not interferon-gamma augmented arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. Treatment of the cells with anti-CD14 and anti-toll-like receptor 4 but not anti-toll-like receptor 2 antibody decreased arginase activity in A. actinomycetemcomitans-lipopolysaccharide-stimulated cells. CONCLUSION: The results of the present study suggest that lipopolysaccharide from A. actinomycetemcomitans via CD14/toll-like receptor 4 complex molecules and the regulatory control of glucocorticoid and cytokines may stimulate arginase activity in RAW264.7 cells.

Viable head and neck tumor spheroids stimulate in vitro autologous monocyte MCP-1 secretion through soluble substances and CD14/lectin-like receptors.

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma (HNSCC) patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro augment their secretion of monocyte chemotactic protein-1 (MCP-1). Presently, the aims of the present work were to study whether the metabolic activity, secreted products and/or specific receptor/ligand on the surface of the F-spheroids and monocytes are necessary for stimulation of the monocyte MCP-1 secretion upon F-spheroid co-culture. Actinomycin D (1 mug/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte MCP-1 co-culture response. Co-culture of monocytes and F-spheroids separated by a semi-permeable membrane showed a decreased, but still present, monocyte MCP-1 co-culture response. Conditioned medium from F-spheroids stimulated allogenous monocytes to secrete MCP-1. The addition of glucose or galactose, but not mannose, to co-cultures partially inhibited the monocyte MCP-1 co-culture response. The addition of anti-CD14 antibody diminished the MCP-1 co-culture response. In conclusion, the monocyte MCP-1 co-culture response is dependent on metabolically active spheroids, secreted stimuli, and is augmented by direct contact with F-spheroids, possibly via lectin-like receptors and the CD14 receptor.

Potentiation of lipopolysaccharide-induced chemokine and adhesion molecule expression in corneal fibroblasts by soluble CD14 or LPS-binding protein.

PURPOSE: The detection of bacterial lipopolysaccharide (LPS) by human cells is facilitated by LPS-binding protein (LBP) and soluble (s)CD14. The effects of these proteins on chemokine release and adhesion molecule expression in cultured human corneal fibroblasts were examined. METHODS: The release of chemokines into culture supernatants and the expression of the intercellular adhesion molecule (ICAM)-1 on the cell surface were determined by enzyme-linked immunosorbent assays. The intracellular abundance of chemokine and ICAM-1 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analyses. The phosphorylation and degradation of IkappaB-alpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. RESULTS: Neither sCD14 nor LBP alone affected the expression of chemokines or ICAM-1 in cultured human corneal fibroblasts. However, sCD14 or LBP enhanced the LPS-induced upregulation of ICAM-1 and the chemokines interleukin-8 and monocyte chemoattractant protein (MCP)-1 in these cells at the protein and mRNA levels. Combined stimulation with LPS and either sCD14 or LBP also induced the phosphorylation and degradation of IkappaB-alpha and the translocation of NF-kappaB from the cytoplasm to the nucleus of corneal fibroblasts. CONCLUSIONS: LBP and sCD14 may play important roles in the defense of the cornea against bacterial infection, by facilitating the detection of LPS by corneal fibroblasts.

Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling.

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. In this study, we tested whether TLR4 signaling promotes a proinflammatory phenotype in human and mouse arterial smooth muscle cells (SMC), characterized by increased cytokine and chemokine synthesis and increased TLR expression. Human arterial SMC were found to express mRNA encoding TLR4 and the TLR4-associated molecules MD-2 and CD14 but not TLR2 mRNA. Mouse aortic SMC, on the other hand, expressed both TLR2 and TLR4 mRNA constitutively. Human SMC derived from the coronary artery, but not those from the pulmonary artery, were found to express cell surface-associated CD14. Low concentrations (ng/ml) of Escherichia coli LPS, the prototypical TLR4 agonist, markedly stimulated extracellular regulated kinase 1/2 (ERK1/2) activity, induced release of monocyte-chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and stimulated IL-1alpha expression in human aortic SMC, and exogenous CD14 enhanced these effects. Expression of a dominant negative form of TLR4 in human SMC attenuated LPS-induced ERK1/2 and MCP-1 release. LPS was a potent inducer of NF-kappaB activity, ERK1/2 phosphorylation, MCP-1 release, and TLR2 mRNA expression in wild-type mice but not in TLR4-signaling deficient mouse aortic SMC. These studies show that TLR4 signaling promotes a proinflammatory phenotype in vascular smooth muscle cells (VSMC) and suggest that VSMC may potentially play an active role in vascular inflammation via the release of chemokines, proinflammatory cytokines, and increased expression of TLR2.

Inhibition of transforming growth factor beta (TGFbeta1) expression and extracellular matrix secretion in human peritoneal mesothelial cells by pcDU6 vector-mediated TGFbeta1 shRNA and by pcDNA3.1(-)-mediated antisense TGFbeta1 RNA.

We investigated the effect of transforming growthfactor beta (TGFbeta1) short hairpin RNA (shRNA) mediated by pcDU6 plasmid on TGFbeta1 expression in human peritoneal mesothelial cells (HPMCs) and compared that effect with the effect of antisense TGFbeta1 RNA. We designed two pairs of oligonucleotides for two selectedfragments of coding sequence containing a 21-nucleotide (nt) TGFbeta1 sequence starting with GGCC. After annealing, double-stranded DNA was formed and separately ligated to plasmid pcDU6 [pcDNA3.1(-) with U6 promoter). The inverted motif contained six spacers and four Ts, which made it possible to form shRNA (TGFbgeta1 shRNA1 and TGFbeta1 shRNA2). We generated recombinant human TGFbeta1 antisense mammalian expression vector, and we isolated HPMCs from human greater omentum by pancreatin disaggregation to establish a stable cell-culture model. We used Lipofectamine 2000 to transfect third-passage HPMCs with plasmid pcDU6 mediating the expression of TGFbeta1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFbeta1 messenger RNA (mRNA). The resulting transfected cells were then stimulated with 4.25% D-glucose and 10 microg/mL lipopolysaccharide (GS+LPS). We used semi-quantitative reverse-transcriptase polymerase chain reaction to detect the expression of TGFbeta1, fibronectin (FN), collagen 1, and plasminogen activator inhibitor type 1 (PAI-1) mRNA by the stimulated cells. The TGFbeta1, FN, and PAI-1 protein levels in the culture supernatant were measured with a sandwich enzyme-linked immunosorbent assay. Expression of TGFbeta1 was significantly upregulated in HPMCs stimulated with GS+LPS (p < 0.01). As compared with control HPMCs in serum-free F12 medium, HPMCs transfected with TGFbeta1 antisense RNA showed inhibited expression of FN, collagen 1, and PAI-1 mRNA (17%, 26%, and 9.6% respectively after 24 hours). Forty-eight hours after transfection, the FN and PAI-I proteins were inhibited by 54.55% and 61.13% respectively (p < 0.05). In the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups, TGFbeta1 expression was obviously downregulated as compared with the GS+LPS group and the pcDU6 void vector group (p < 0.01). No significant difference was observed between the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups (p > 0.05). No significant difference was observed between the pcDNA3.1(-) vector-mediated antisense RNA group and the pcDU6 void vector group (p > 0.05). The expression of TGFbeta1 in pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups was obviously downregulated as compared with the pcDNA3.1(-) plasmid vector-mediated antisense RNA group (p < 0.01). In HPMCs stimulated with GS+LPS, pcDU6 plasmid vector-mediated shRNA can significantly inhibit the induced expression of TGFbeta1. These results suggest the possible application of pcDU6 plasmid vector-mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.

Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

Differences in LPS-induced activation of bronchial epithelial cells (BEAS-2B) and type II-like pneumocytes (A-549).

Lipopolysaccharide (LPS) as a major component of the outer membrane of gram-negative bacteria stimulates various cells to initiate a signalling cascade which ultimately leads to cell activation and expression of immunoregulatory or inflammatory cytokines. The human respiratory epithelium is an important environmental interface, but differences in LPS-induced cell activation between bronchial and alveolar epithelial cells have not yet been investigated in detail. First, the expression of Toll-like receptors (TLRs), as pattern-recognition receptors, was investigated for the bronchial epithelial cells and type II-like pneumocytes, demonstrating that they fulfil the prerequisites for LPS signalling. Thereafter, the effects of LPS, soluble CD14 (sCD14) and LPS-binding protein (LBP) on the release of interleukin-6 (IL-6) and IL-8 were studied. In the presence of LPS, sCD14 induced a significant and concentration-dependent cytokine release in type II-like pneumocytes, whereas the response of bronchial epithelial cells to sCD14 stimulation was low, implicating sCD14-independent activation mechanisms. Furthermore, LBP revealed inhibitory effects on the activation of alveolar epithelial cells, which may represent a novel local defence mechanism during gram-negative infection. We conclude that distinct pathways exist for LPS-induced activation of bronchial and alveolar epithelial cells.

Recombinant bovine soluble CD14 sensitizes the mammary gland to lipopolysaccharide.

Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis. It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis. Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface. In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody. Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk. In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection. These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo. Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.

Lactoferrin inhibits the lipopolysaccharide-induced expression and proteoglycan-binding ability of interleukin-8 in human endothelial cells.

Interleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complex Escherichia coli 055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans.

Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4-mediated signaling.

Toll-like receptors (TLRs) have recently been identified as fundamental components of the innate immune response to bacterial pathogens. We investigated the role of TLR signaling in immune defense of the mucosal epithelial cells of the lower female genital tract. This site provides first line defense against microbial pathogens while remaining tolerant to a complex biosystem of resident microbiota. Epithelial cells derived from normal human vagina, ectocervix, and endocervix expressed mRNA for TLR1, -2, -3, -5, and -6. However, they failed to express TLR4 as well as MD2, two essential components of the receptor complex for LPS in phagocytes and endothelial cells. Consistent with this, endocervical epithelial cells were unresponsive to protein-free preparations of lipooligosaccharide from Neisseria gonorrhoeae and LPS from Escherichia coli. However, they were capable of responding to whole Gram-negative bacteria and bacterial lysates, as demonstrated by NF-kappaB activation and proinflammatory cytokine production. The presence of soluble CD14, a high-affinity receptor for LPS and other bacterial ligands, enhanced the sensitivity of genital tract epithelial cells to both low and high concentrations of bacteria, suggesting that soluble CD14 can act as a coreceptor for non-TLR4 ligands. These data demonstrate that the response to N. gonorrhoeae and other Gram-negative bacteria at the mucosal surface of the female genital tract occurs in the absence of endotoxin recognition and TLR4-mediated signaling.

Regulation of bovine E-selectin expression by recombinant tumor necrosis factor alpha and lipopolysaccharide.

Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue. Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics. Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) or Escherichia coli lipopolysaccharide (LPS). E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines. Surface expression of E-selectin was measured by flow cytometry. Unstimulated BAECs expressed minimum or no E-selectin on the surface. A low number of endothelial cells expressed surface E-selectin as early as 1h post-stimulation and surface expression was sustained after both stimuli for 24-72h. Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72h without returning to baseline values. Mean fluorescence intensity following stimulation with TNF-alpha increased slightly between 0 and 72h. The pattern of mRNA expression differed between stimuli. LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h. Conversely, BAECs stimulated with rbTNF-alpha expressed significantly (p pound 0.05) higher amounts of mRNA at 1h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h. To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18-72h. Culture supernatants from rbTNF-alpha-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA. Unstimulated culture supernatants contained little or no E-selectin. Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-alpha-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.

Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.